Peripancreatic fat necrosis worsens acute pancreatitis independent of pancreatic necrosis via unsaturated fatty acids increased in human pancreatic necrosis collections

Abstract

Background and aims: Peripancreatic fat necrosis occurs frequently in necrotising pancreatitis. Distinguishing markers from mediators of severe acute pancreatitis (SAP) is important since targeting mediators may improve outcomes. We evaluated potential agents in human pancreatic necrotic collections (NCs), pseudocysts (PCs) and pancreatic cystic neoplasms and used pancreatic acini, peripheral blood mononuclear cells (PBMC) and an acute pancreatitis (AP) model to determine SAP mediators.

Methods: We measured acinar and PBMC injury induced by agents increased in NCs and PCs. Outcomes of caerulein pancreatitis were studied in lean rats coadministered interleukin (IL)-1β and keratinocyte chemoattractant/growth-regulated oncogene, triolein alone or with the lipase inhibitor orlistat.

Results: NCs had higher fatty acids, IL-8 and IL-1β versus other fluids. Lipolysis of unsaturated triglyceride and resulting unsaturated fatty acids (UFA) oleic and linoleic acids induced necro-apoptosis at less than half the concentration in NCs but other agents did not do so at more than two times these concentrations. Cytokine coadministration resulted in higher pancreatic and lung inflammation than caerulein alone, but only triolein coadministration caused peripancreatic fat stranding, higher cytokines, UFAs, multisystem organ failure (MSOF) and mortality in 97% animals, which were prevented by orlistat.

Conclusions: UFAs, IL-1β and IL-8 are elevated in NCs. However, UFAs generated via peripancreatic fat lipolysis causes worse inflammation and MSOF, converting mild AP to SAP.

Keywords: OBESITY; PANCREATITIS.

Figure 1

Necrotic collections (NCs) occur in obese patients and are enriched in DNA and unsaturated fatty acids (UFAs). (A) Table showing biometric data, disease duration at the time of intervention and aetiology of pancreatic fluid collections. Patients with pancreatic cystic neoplasms (PCNs) were older than and those with NCs had higher body mass index (BMI) than those with pseudocysts (PCs). p Values for age and BMI (one-way analysis of variance) and gender (two-tailed Fishers exact test) are mentioned. * indicates groups significantly different (p<0.05) from each other. Box plots showing (B) DNA, (C) proportion of UFAs, (D) amounts of UFA, (E) non-esterified fatty acid (NEFA), (F) SFAs in NC, PC and PCN. * indicates groups significantly different (p<0.05), and ‘NS’ those not significantly different from each other.

Figure 2

Necrotic collections (NCs) are enriched in interleukin (IL)-1β, IL-8 and resistin. Box plots showing (A) IL-1β, (B) IL-8 and (C) resistin levels in log scale being elevated in NC and pseudocysts (PC) compared with pancreatic cystic neoplasms (PCN). * indicates groups significantly different (p<0.05), and ‘NS’ those not significantly different from each other.

Figure 3

Unsaturated fatty acids generated via triglyceride lipolysis induce acinar cell death: (A) glycerol concentrations in medium from acini incubated with saturated (glyceryl tripalmitate (GTP)) and unsaturated triglycerides (glyceryl trilinoleate (GTL)), glyceryl trioleate (GTO)) are reduced by orlistat. Only GTL and GTO cause cell injury quantified as (B)% lactate dehydrogenase (LDH) leakage, which is prevented by orlistat (50 μM). Acinar necrosis measured at 5 h as %LDH leakage (C) or as propidium iodide uptake (D) induced by various agents elevated in necrotic collections (NCs) and pseudocysts. Only oleic (OA) and linoleic (LA) acids induced necrosis at half their concentrations in NCs. Palmitic acid, stearic acid (SA) and inflammatory cytokines (10 ng/mL each) did not cause necrosis. These are more than two times the concentrations in NCs. * indicates groups significantly different (p<0.05) from control, and † indicates significant reduction of measured parameter upon lipase inhibition by orlistat versus without orlistat. Numbers on X-axis represent micromolar amounts.

Figure 4

During caerulein pancreatitis, coadministration of interleukin (IL)-1β with keratinocyte chemoattractant (KC)/growth-regulated oncogene (GRO), or intraperitoneal triolein alone or with orlistat does not affect pancreatitis initiation or cause pancreatic necrosis: serum amylase (A) and lipase (B) were similarly increased more than threefold above normal in all groups with caerulein pancreatitis at 1 day. Fat stranding in the peritoneal cavity (C) and surface of pancreata (D) in animals coadministered triolein with caerulein (CER) was prevented by orlistat, with remnant unhydrolysed triolein appearing as a thick whitish liquid coating. (E) Histologically there was no evidence of necrosis in any group. Please note ductal metaplasia-like appearance in survivors up to 3 days.

Figure 5

Interleukin (IL)-1β+keratinocyte chemoattractant (KC)/growth-regulated oncogene (GRO) and triolein coadministration results in worse local and systemic inflammation during caerulein (CER) pancreatitis. Immunohistochemistry images for myeloperoxidase (MPO) in the pancreas (A1–5) and lungs (B1–5), with each group mentioned at the bottom of the image. While caerulein pancreatitis increased MPO-positive cells compared with controls (A2, A6*), IL-1β+KC/GRO coadministration caused a further increase (A3, A6 †), compared with caerulein alone. Systemically, in the lungs, IL-1β+KC/GRO coadministration (B3) and triolein coadministration (B4) significantly increased MPO-positive cells compared with caerulein pancreatitis alone (B2) or other groups (B1,4,5). Orlistat treatment significantly reduced (B6, †) the lung MPO increased noted in the CER+ triolein group.

Figure 6

Lipolysis of triolein unlike interleukin (IL)-1β+keratinocyte chemoattractant (KC)/growth-regulated oncogene (GRO) coadministration results in a worse cytokine response and mortality in caerulein (CER) pancreatitis. (A) Kaplan–Meier curves showing time course of mortality in the triolein coadministered group (red), which is prevented by orlistat (green) and is absent in other groups. Number per group is mentioned in parenthesis. Serum oleate (B), IL-1β (C) and KC/GRO (D) are significantly increased in the acute pancreatitis (AP) group coadministered triolein and reduced by orlistat. Multiple comparisons were done using one-way analysis of variance. Significant (p<0.05) difference from controls is denoted with an *; † over the CER+triolein+orlistat group indicates significant difference from CER+triolein.

Figure 7

Lipolysis of triolein unlike interleukin (IL)-1β+keratinocyte chemoattractant (KC)/growth-regulated oncogene (GRO) coadministration results in lung injury during caerulein (CER) pancreatitis: images of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of lungs (A–E) with each group mentioned at the bottom of the image. There was significantly more positive staining in the lungs (D) of CER+triolein group (*), which is prevented by orlistat (E and F), depicted as †. Bronchoalveolar lavage (BAL) fluid was analysed for fluorescein isothiocyanate (FITC) fluorescence (G), lactate dehydrogenase (LDH; H), total protein (I) or stained with propidium iodide, and the fluorescent cells were counted (J). Significant (p<0.05) difference from controls is denoted with an *; † over the CER+triolein+orlistat group indicates significant difference from CER +triolein.

Figure 8

Lipolysis of triolein unlike interleukin (IL)-1β+keratinocyte chemoattractant (KC)/growth-regulated oncogene (GRO) coadministration results in necro-apoptotic injury and renal failure during caerulein (CER) pancreatitis: images of terminal deoxynucleotidyl transferase dUTP nick end labelling (A–E) and immunohistochemistry for kidney injury molecule-1 (A1–E1) in kidneys, with each group mentioned above the image. There was positive staining in the CER+triolein group, sloughing of renal tubular cells (black boxes) along with renal failure evidenced by an increase in serum blood urea nitrogen (BUN) (F, *), which is prevented by orlistat depicted as †. Rat peripheral blood mononuclear cells from the control group (G), those treated with IL-1b+KC/GRO (200 ng/mL each for 30 min; H), 10 μM oleic acid (OA) for 10 min (I) or 30 min (J) were stained for annexin V and propidium iodide (PI) and analysed by flow cytometry. OA significantly increased (*) early apoptosis at 10 min (K), which progressed to predominantly necro-apoptosis by 30 min (L) with progressively increasing necrosis (M).